T cells are a type of lymphocyte that is involved in immune system regulation and function, particularly those that are driven in response to a specific antigen. Immune cells such as T cells can be identified by the combination of cell markers they express. For example, T cells express cluster of differentiation (CD) 3, which is part of the T cell receptor (TCR) complex. Further, a distinct population of T cells express CD4 (the so-called “helper” T cells), while another distinct population of T cells express CD8 (the so-called “cytotoxic” T cells). The expression marker CD25 is a marker of activation on a number of types of cells, and it is particularly upregulated following stimulation of a cell, notably following antigen stimulation. Activated and memory CD4+ T cells co-express CD4 and CD25.
Regulatory T cells (Treg), also known as “suppressor” T cells, are a specialised subpopulation of T cells that function to suppress activation of the immune system, thereby maintaining immune system homeostasis and tolerance to self-antigens. Treg are accordingly of fundamental importance in suppressing various immune and autoimmune responses. Treg can be identified by their suppressive function as well as by co-expression of T cell markers, for example, CD4, the activation marker CD25 (the α chain of the IL-2 receptor) and the transcription factor Foxp3.
Several different Treg subsets have been described (1). A naturally occurring, distinct population of CD4+CD25+Foxp3+ Treg known as natural Treg (nTreg) develop in the thymus and are present in healthy individuals from birth. The specificity of the T cell receptor (TCR) of nTreg is mainly self-reactive. Additionally, a population of CD4+CD25+Foxp3+ Treg can be induced in vivo in the periphery under various conditions, such as during certain defined conditions of antigen presentation and cytokine stimulation, and can induce tolerance (reviewed in (2)). Different subsets of inducible Treg have been reported, including T regulatory type 1 (TR1) cells, which produce high levels of interleukin-1 0 (IL-1 0) (3), a cytokine that has anti-inflammatory actions and facilitates suppression of the antigen presentation capacity of antigen presenting cells. Additionally, a subset of CD4+CD25+Poxp3+ Treg can be induced in vitro from CD4+CD25− T cells in the presence of transforming growth factor-13 (TGF13) (4).
Data generated in several animal models indicates that adoptive transfer of Treg can prevent or cure T cell mediated diseases, autoimmune diseases and allograft rejection, by restoring immune tolerance to self-antigens or alloantigens (5-8). Absent or defective Treg function has been correlated with autoimmunity in humans, and the presence of Treg has been associated with immunological tolerance. Defective Treg in peripheral blood from patients with multiple sclerosis, type-1 diabetes, psoriasis, myasthenia gravis, rheumatoid and juvenile idiopathic arthritis have been described (reviewed in (9)). Defective Treg have also been reported in genetic diseases like immunodysregulation, polyendocrinopathy and enteropathy X-linked syndrome (IPEX)(10), Wiskott-Aldrich syndrome (WAS) (11), autoimmune polyglandular syndrome (APS) type 2 (12) and autoimmune lymphoproliferative syndrome (ALPS) (13).
Accordingly, detecting the presence or absence of Treg cells specific for a particular antigen of interest (ie target antigen) may facilitate diagnosis or assessment of immunological conditions or diseases. Additionally, Treg-based therapy may provide an effective means to treat diseases where suppression of the immune response may be beneficial such as autoimmune diseases, allergic diseases, immunoinflammatory diseases and T cell mediated diseases including genetic diseases. Treg-based therapy may also be useful in suppressing graft rejection, including suppression of graft-versus-host disease (GVHD) after haematopoietic stem cell transplantation (HSCT) (14-16). The use of Treg in the clinic to treat a number of conditions or diseases is currently under consideration by several groups (reviewed in (2)). However, there are a number of issues that are currently impeding Treg therapy.
First, accurate identification of viable Treg using the CD4, CD25 and Foxp3 markers is problematical. Both antigen-experienced conventional effector CD4+ T cells and CD4+ Treg both express CD25. Further, detection of Treg using Foxp3 antibodies requires fixation and permeabilisation of the cells, so the technique cannot be used to isolate viable Treg populations for functional studies or ex vivo expansion as a prelude to therapeutic administration. However, WO 2007/014420 describes a method of detecting viable Treg using the cell marker CD127, the α chain of the interleukin (IL)-7 receptor, in combination with CD4 and CD25. For example, it was shown that CD4+CD25+CD127lo expression is indicative of a regulatory T cell or a population of regulatory T cells (17). More recently, CD39 and CD73, two ectoenzymes that generate adenosine resulting in suppression of T cell responses, have been reported as useful markers for Treg (18-19).
Secondly, Treg are present in low numbers in the circulation. While methods exist to induce and expand Treg ex vivo, Treg have a range of antigen specificities, and in order to suppress a particular inappropriate immune response in certain conditions or diseases (eg autoimmune diseases, allergic diseases, immunoinflammatory diseases, infectious diseases, allograft rejection, and T cell mediated diseases including genetic diseases), Treg need to specifically recognise the antigen involved in the response. However, methods of detecting the antigen specificity of Treg have not previously been described.
WO 2007/106939 describes a highly sensitive flow cytometric assay which is capable of identifying antigen specific effector (conventional) CD4+ or CDS+ T cells using antibodies directed to the cell marker CD25, in combination with antibodies directed to one or more of CD134 (also known as OX40) and, CD137 (also known as 4-1BB) following exposure to the target antigen. For example, following in vitro exposure to a particular target antigen in whole blood, CD4+ T cells that were specific for that antigen were shown to co-express CD25 and CD134. However, Treg were not thought to be identified by this method. CD134 is not expressed in CD4+CD25+ Treg cells isolated from human blood (32). Further, a recent report indicates that stimulation of CD134 on Treg by an anti-CD134 antibody down regulated Foxp3 expression and reduced Treg function in mice (48).
The present applicant has found that antigen-specific CD4+ Treg can be identified by detecting cells expressing the combination of CD4, CD25, CD134 and one or more Treg markers, such as CD39, CD127 (wherein CD127 expression is preferably CD127lo) and Foxp3; and further, that antigen-specific CD8+ Treg can be identified by the expression of the combination of CD8, CD25, CD137 and one or more Treg cell markers, such as CD39, CD127 (wherein CD127 expression is preferably) CD127lo) and Foxp3. Surprisingly, the present applicant has also found that viable antigen-specific Treg can be identified and/or isolated using combinations of the cell markers CD4, CD8, CD25, CD39, CD45RO, CD45RA, CD127, CD134 and CD137. Moreover, it was found that using such combinations of these cell markers provides a means to isolate Treg of high purity, which may be ex vivo expanded, for use in, for example, Treg-based therapy.